Process for preparing L-amino acids using improved strains of the enterobacteriaceae family

ABSTRACT

The invention relates to a process for preparing L-amino acids by fermenting recombinant microorganisms of the Enterobacteriaceae family, characterized in that a) the desired L-amino acid-producing microorganisms, in which the yjcG-ORF, or nucleotide sequences or alleles encoding the gene product, is/are enhanced, in particular overexpressed, is cultured in a medium under conditions under which the desired L-amino acid is accumulated in the medium or in the cells, and b) the desired L-amino acid is isolated, with, where appropriate, constituents of the fermentation broth, and/or the biomass remaining in its/their entirety or in portions (from ≧0 to 100%) in the isolated product or being removed completely.

This invention relates to a process for preparing L-amino acids, inparticular L-threonine, using recombinant microorganisms strains of theEnterobacteriaceae family in which the open reading frame (ORF)designated yjcG is enhanced, in particular overexpressed, and to saidmicroorganisms.

PRIOR ART

L-Amino acids, in particular L-threonine, are used in human medicine andin the pharmaceutical industry, in the foodstuff industry and, veryparticularly, in animal nutrition.

It is known that L-amino acids can be prepared by fermentingEnterobacteriaceae strains, in particular Escherichia coli (E. coli) andSerratia marcescens. Because of the great importance, efforts arecontinually being made to improve the preparation methods.Methodological improvements can concern measures relating tofermentation technology, such as stirring or supplying with oxygen, orthe composition of the nutrient media, such as the sugar concentrationduring the fermentation, or the working-up to the product form, forexample by means of ion exchange chromatography, or the intrinsicperformance properties of the microorganism itself.

Methods of mutagenesis, selection and mutant choice are used forimproving the performance properties of these microorganisms. Thisthereby results in strains which are resistant to antimetabolites, suchas the threonine analog α-amino-β-hydroxyvaleric acid (AHV), orauxotrophic for metabolites of regulatory importance and produce L-aminoacids such as L-threonine.

For a number of years now, recombinant DNA methods have also been usedfor improving L-amino acid-producing strains of the Enterobacteriaceaefamily by amplifying individual amino acid biosynthesis genes andinvestigating the effect on production. Compiled information on the cellbiology and molecular biology of Escherichia coli and Salmonella can befound in Neidhardt (ed): Escherichia coli and Salmonella, Cellular andMolecular Biology, 2^(nd) edition, ASM Press, Washington, D.C., USA,(1996).

OBJECT OF THE INVENTION

The inventors have set the object of providing novel measures forimproving the fermentative preparation of L-amino acids, in particularL-threonine.

DESCRIPTION OF THE INVENTION

The invention relates to recombinant microorganisms of theEnterobacteriaceae family which contain an enhanced or overexpressedopen reading frame yjcG, which encodes a polypeptide which is annotatedas being acetate permease, or nucleotide sequences encoding its geneproduct and which display an improved ability to form and accumulateL-amino acids, in particular L-threonine.

In each case, the microorganisms which are not recombinant for theyjcG-ORF, which do not contain any enhanced yjcG-ORF, and on which themeasures of the invention are performed are used as the starting pointfor the comparison.

These recombinant microorganisms include, in particular, microorganismsof the Enterobacteriaceae family in which a polynucleotide which encodesa polypeptide whose amino acid sequence is at least 90%, in particularat least 95%, preferably at least 98%, are at least 99%, particularlypreferably 99.8% and very particularly preferably 100%, identical to anamino acid sequence selected from the group SEQ ID No. 2, SEQ ID No. 4and SEQ ID No. 6 is enhanced.

Said microorganisms contain enhanced or overexpressed polynucleotidesselected from the group:

-   a) polynucleotide having a nucleotide sequence, selected from SEQ ID    No. 1, SEQ ID No. 3 and SEQ ID No. 5 and the nucleotide sequences    complementary thereto;-   b) polynucleotide having a nucleotide sequence which corresponds to    SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 5 within the limits of the    degeneracy of the genetic code;-   c) polynucleotide sequence having a sequence which hybridizes, under    stringent conditions, with the sequence which is complementary to    the sequence SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 5, with the    stringent conditions preferably being achieved by means of a washing    step in which the temperature extends over a range of from 64° C. to    68° C. and the salt concentration of the buffer extends over a range    of from 2×SSC to 0.1×SSC;-   d) polynucleotide having a sequence SEQ ID No. 1, SEQ ID No. 3 or    SEQ ID No. 5 which contains functionally neutral sense mutants,    with the polynucleotides preferably encoding an acetate permease.

The invention also relates to a process for fermentatively preparingL-amino acids, in particular L-threonine, using recombinantmicroorganisms of the Enterobacteriaceae family which, in particular,already produce L-amino acids and in which at least the open readingframe (ORF) having the designation yjcG, or nucleotide sequencesencoding its gene product, is or are enhanced.

Preference is given to using the microorganisms which are described.

When L-amino acids or amino acids are mentioned in that which follows,this thereby means one or more amino acids, including their salts,selected from the group L-asparagine, L-threonine, L-serine,L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine,L-proline, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine,L-histidine, L-lysine, L-tryptophan; L-arginine and L-homoserine.L-threonine is particularly preferred.

In this connection, the term “enhancement” describes the increase, in amicroorganism, of the intracellular activity or concentration of one ormore enzymes or proteins which are encoded by the corresponding DNA,with, for example, the copy number of the gene or genes, or of the ORFor ORFS, being increased by at least one (1) copy, use being made of astrong promoter operatively linked to the gene or of a gene or allele orORF which encodes a corresponding enzyme or protein having a highactivity, and, where appropriate, these measures being combined.

A segment of a nucleotide sequence which encodes, or can encode, aprotein and/or a polypeptide or ribonucleic acid to which the prior artis unable to assign any function is designated an open reading frame(ORF). After a function has been assigned to the nucleotide sequencesegment in question, this segment is generally referred to as being agene. Alleles are generally understood as being alternative forms of agiven gene. The forms are distinguished by differences in the nucleotidesequence.

In general, the protein, or the ribonucleic acid, encoded by anucleotide sequence, i.e. an ORF, a gene or an allele, is designated agene product.

The enhancement measures, in particular overexpression, generallyincrease the activity or concentration of the corresponding protein byat least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%,maximally up to 1000% or 2000%, based on that of the wild-type proteinor on the activity or concentration of the protein in the parent strainor microorganism which is not recombinant for the corresponding enzymeor protein. The non-recombinant microorganism or parent strain isunderstood as being the microorganism on which the measures according tothe invention are performed.

The invention relates to a process for preparing L-amino acids byfermenting recombinant microorganisms of the Enterobacteriaceae family,characterized in that

-   -   a) the desired L-amino acid-producing microorganisms, in which        the open reading frame yjcG, or nucleotide sequences or alleles        encoding the gene product, is/are enhanced, in particular        overexpressed, are cultured in a medium under conditions under        which the desired L-amino acid is accumulated in the medium or        in the cells, and    -   b) the desired L-amino acid is isolated, with, where        appropriate, the fermentation broth constituents and/or the        biomass remaining in its/their entirety or in portions (from ≧0        to 100%) in the isolated product or being removed completely.

The microorganisms which have an enhanced or overexpressed open readingframe (ORF) designated yjcG, and which are in particular recombinant,are likewise part of the subject matter of the present invention, canproduce L-amino acids from glucose, sucrose, lactose, fructose, maltose,molasses, where appropriate starch and where appropriate cellulose orfrom glycerol and ethanol. The microorganisms are representatives of theEnterobacteriaceae family and are selected from the genera Escherichia,Erwinia, Providencia and Serratia. The genera Escherichia and Serratiaare preferred. The species Escherichia coli may be mentioned, inparticular, in the case of the genus Escherichia while the speciesSerratia marcescens may be mentioned, in particular, in connection withthe genus Serratia.

In general, recombinant microorganisms are generated by means oftransformation, transduction or conjugation, or a combination of thesemethods, with a vector which contains the desired ORF, the desired gene,an allele of this ORF or gene, or parts thereof, and/or a promoter whichpotentiates the expression of the ORF or gene. This promoter can be thepromoter which has been produced by enhancing mutation from theendogenous regulatory sequence located upstream of the gene or ORF;alternatively, an efficient promotor has been fused to the gene or ORF.

Examples of strains of the genus Escherichia, in particular of thespecies Escherichia coli, which are suitable as parent strain, whichproduce L-threonine, in particular, and which are to be mentioned are:

-   -   Escherichia coli H4581 (EP 0 301 572)    -   Escherichia coli KY10935 (Bioscience Biotechnology and        Biochemistry 61(11):1877-1882 (1997)    -   Escherichia coli VNIIgenetica MG442 (U.S. Pat. No. 4,278,765)    -   Escherichia coli VNIIgenetica M1 (U.S. Pat. No. 4,321,325)    -   Escherichia coli VNIIgenetica 472T23 (U.S. Pat. No. 5,631,157)    -   Escherichia coli BKIIM B-399.6 (U.S. Pat. No. 5,175,107)    -   Escherichia coli cat 13 (WO 98/04715)    -   Escherichia coli KCCM-10132 (WO 00/09660)

Examples of L-threonine-producing strains of the genus Serratia, inparticular of the species Serratia marcescens, which are suitable asparent strain and which are to be mentioned are:

-   -   Serratia marcescens HNr21 (Applied and Environmental        Microbiology 38(6): 1045-1051 (1979))    -   Serratia marcescens TLrl56 (Gene 57(2-3): 151-158 (1987))    -   Serratia marcescens T-2000 (Applied Biochemistry and        Biotechnology 37(3): 255-265 (1992))

L-Threonine-producing strains of the Enterobacteriaceae familypreferably possess, inter alia, one or more of the genetic or phenotypicfeatures selected from the group: resistance to α-amino-β-hydroxyvalericacid, resistance to thialysine, resistance to ethionine, resistance toα-methylserine, resistance to diaminosuccinic acid, resistance toα-aminobutyric acid, resistance to borrelidin, resistance tocyclopentanecarboxylic acid, resistance to rifampicin, resistance tovaline analogs such as valine hydroxamate, resistance to purine analogs,such as 6-dimethylaminopurine, requirement for L-methionine, possiblepartial and compensatable requirement for L-isoleucine, requirement formesodiaminopimelic acid, auxotrophy in regard to threonine-containingdipeptides, resistance to L-threonine, resistance to threonineraffinate, resistance to L-homoserine, resistance to L-lysine,resistance to L-methionine, resistance to L-glutamic acid, resistance toL-aspartate, resistance to L-leucine, resistance to L-phenylalanine,resistance to L-serine, resistance to L-cysteine, resistance toL-valine, sensitivity to fluoropyruvate, defective threoninedehydrogenase, possible ability to utilize sucrose, enhancement of thethreonine operon, enhancement of homoserine dehydrogenase I-aspartatekinase I, preferably of the feedback-resistant form, enhancement ofhomoserine kinase, enhancement of threonine synthase, enhancement ofaspartate kinase, possibly of the feedback-resistant form, enhancementof aspartate semialdehyde dehydrogenase, enhancement ofphosphoenolpyruvate carboxylase, possibly of the feedback-resistantform, enhancement of phospho-enolpyruvate synthase, enhancement oftranshydrogenase, enhancement of the RhtB gene product, enhancement ofthe RhtC gene product, enhancement of the YfiK gene product, enhancementof a pyruvate carboxylase and attentuation of acetic acid formation.

It has been found that, following overexpression of the gene or the openreading frame (ORF) yjcG, or its alleles, microorganisms of theEnterobacteriaceae family display an improved ability to form andaccumulate L-amino acids, in particular L-threonine.

The nucleotide sequences of the Escherichia coli genes or open readingframes (ORFs) belong to the prior art and can be obtained from theEscherichia coli genome sequence published by Blattner et al. (Science277: 1453-1462 (1997)). It is known that endogenous enzymes (methionineaminipeptidase) are able to cleave off the N-terminal amino acidmethionine.

The nucleotide sequences for the yjcG-ORF from Salmonella typhimirium(Accession No.: NC_(—)003197 (Region 4511508-4513157)) and Shigellaflexneri (Accession No.: NC_(—)004337 (Region 4293843-4295492)), whichlikewise belong to the Enterobacteriaceae family, have also beendisclosed.

The yjcG-ORF of Escherichia coli K12 is described, inter alia, by thefollowing data:

Gimenez et al. (Journal of Microbiology 185(21): 6448-55 (2003))describe the Escherichia coli open reading frame yjcG, which encodes aprotein having the function of an acetate permease from the solute:sodium symporter (SSS) family, and propose the gene name actP; the geneis cotranscribed with acs, which encodes an acetyl coenzyme Asynthetase; the permease is highly specific for short-chain aliphaticmonocarboxylates and, in addition to acetate, also transports glycolatein a quantity sufficient for growth, as well as small carboxylates suchas propionate.

Accession No.: NC000913 (Region 4281276-4282925)

Alternative gene name: b4067, actP

The nucleic acid sequences can be obtained from the databases belongingto the National Center for Biotechnology Information (NCBI) of theNational Library of Medicine (Bethesda, Md., USA), the nucleic acidsequence database of the European Molecular Biology Laboratories (EMBL,Heidelberg, Germany or Cambridge, UK) or the Japanese DNA database(DDBJ, Mishima, Japan).

For the sake of greater clarity, the known nucleotide sequence for theyjcG-ORF of Escherichia coli is depicted under SEQ ID No. 1 and theknown sequences for the yjcG-ORF of Salmonella typhimurium or Shigellaflexneri under SEQ ID No. 3 and, respectively, SEQ ID No. 5. Theproteins encoded by these reading frames are depicted as SEQ ID No. 2,SEQ ID No. 4 and SEQ ID No. 6.

The open reading frames described in the passages indicated can be usedin accordance with the invention. In addition, it is possible to usealleles of the genes or open reading frames, which result from thedegeneracy of the genetic code or as a consequence of functionallyneutral sense mutations. Preference is given to using endogenous genesor endogenous open reading frames.

“Endogenous genes” or “endogenous nucleotide sequences” are understoodas being the genes or open reading frames or alleles or nucleotidesequences which are present in a species population.

The alleles of the yjcG-ORF, which contain functionally neutral sensemutations, include, inter alia, those which lead to at most 60 or to atmost 50 or to at most 40 or to at most 30 or to at most 20, preferablyto at most 10 or to at most 5, very particularly preferably to at most 3or to at most 2, or to at least one, conservative amino acidsubstitution in the protein which they encode.

In the case of the aromatic amino acids, the substitutions are said tobe conservative when phenylalanine, tryptophan and tyrosine aresubstituted for each other. In the case of the hydrophobic amino acids,the substitutions are said to be conservative when leucine, isoleucineand valine are substituted for each other. In the case of the polaramino acids, the substitutions are said to be conservative whenglutamine and asparagine are substituted for each other. In the case ofthe basic amino acids, the substitutions are said to be conservativewhen arginine, lysine and histidine are substituted for each other. Inthe case of the acid amino acids, the substitutions are said to beconservative when aspartic acid and glutamic acid are substituted foreach other. In the case of the hydroxyl group-containing amino acids,the substitutions are said to be conservative when serine and threonineare substituted for each other.

In the same way, it is also possible to use nucleotide sequences whichencode variants of said proteins, which variants additionally contain anextension or truncation by at least one (1) amino acid at the N terminusor C terminus. This extension or truncation amounts to not more than 60,50, 40, 30, 20, 10, 5, 3 or 2 amino acids or amino acid residues.

The suitable alleles also include those which encode proteins in whichat least one (1) amino acid has been inserted or deleted. The maximumnumber of such changes, termed indels, can affect 2, 3, 5, 10, 20, butin no case more than 30, amino acids.

The suitable alleles furthermore include those which can be obtained bymeans of hybridization, in particular under stringent conditions, usingSEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 5 or parts thereof, inparticular the coding regions or the sequences which are complementarythereto.

The skilled person finds instructions for identifying DNA sequences bymeans of hybridization in, inter alia, the manual “The DIG System UsersGuide for Filter Hybridization” supplied by Boehringer Mannheim GmbH(Mannheim, Germany, 1993) and Liebl et al. (International Journal ofSystematic Bacteriology 41: 255-260 (1991)). The hybridization takesplace under stringent conditions, that is the only hybrids formed arethose in which the probe and target sequence, i.e. the polynucleotidestreated with the probe, are at least 80% identical. It is known that thestringency of the hybridization, including the washing steps, isinfluenced and/or determined by varying the buffer composition, thetemperature and the salt concentration. In general, the hybridizationreaction is carried out at a stringency which is relatively low ascompared with that of the washing steps (Hybaid Hybridization Guide,Hybaid Limited, Teddington, UK, 1996).

For example, a buffer corresponding to 5×SSC buffer can be used for thehybridization reaction at a temperature of approx. 50° C.-68° C. Underthese conditions, probes can also hybridize with polynucleotides whichpossess less than 70% identity with the sequence of the probe. Thesehybrids are less stable and are removed by washing under stringentconditions. This can be achieved, for example, by lowering the saltconcentration down to 2×SSC and, where appropriate, subsequently to0.5×SSC (The DIG System User's Guide for Filter Hybridization,Boehringer Mannheim, Mannheim, Germany, 1995) with the temperature beingadjusted to approx. 50° C.-68° C., approx. 52° C.-68° C., approx. 54°C.-68° C., approx. 56° C.-68° C., approx. 58° C.-68° C., approx. 60°C.-68° C., approx. 62° C.-68° C., approx. 64° C.-68° C., approx. 66°C.-68° C. Temperature ranges of approx. 64° C.-68° C. or approx. 66°C.-68° C. are preferred. It is possible, where appropriate, to lower thesalt concentration down to a concentration corresponding to 0.2×SSC or0.1×SSC. By means of increasing the hybridization temperature stepwise,in steps of approx. 1-2° C., from 50° C. to 68° C., it is possible toisolate polynucleotide fragments which, for example, possess at least80%, or at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%, identity with the sequence of the probe employed or with thenucleotide sequences shown in SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No.5. Additional instructions for the hybridization can be obtainedcommercially in the form of what are termed kits (e.g. DIG Easy Hyb fromRoche Diagnostics GmbH, Mannheim, Germany, Catalog No. 1603558).

In order to achieve enhancement, it is possible, for example, toincrease the expression of the genes or open reading frames or allelesor to increase the catalytic properties of the protein. Both measurescan be combined, where appropriate.

In order to achieve overexpression, the copy number of the correspondinggenes or open reading frames can be increased, for example, or thepromoter region and regulatory region or the ribosome binding site whichis located upstream of the structural gene can be mutated. Expressioncassettes which are incorporated upstream of the structural gene act inthe same manner. It is also possible to increase expression during thecourse of the fermentative L-threonine production through induciblepromoters; in addition, using promoters for gene expression whichpermits a different chronological gene expression can also beadvantageous. At the level of the translational regulation of geneexpression, it is possible to increase the frequency of initiation(binding of the ribosome to the mRNA) or the rate of elongation(elongation phase). Expression is likewise improved by means of measuresfor extending the lifespan of the mRNA. Furthermore, the enzyme activityis also enhanced by preventing the enzyme protein from being brokendown. The ORFs, genes or gene constructs can either be present inplasmids having different copy numbers or be integrated, and amplified,in the chromosome. Alternatively, overexpression of the genes concernedcan also be achieved by altering the composition of the media and theconduct of the culture.

Methods for overexpression are adequately described in the prior art,for example in Makrides et al. (Microbiological Reviews 60 (3), 512-538(1996)). Using vectors increases the copy number by at least one (1)copy. The vectors used can be plasmids as described, for example, inU.S. Pat. No. 5,538,873. The vectors used can also be phages, forexample phage Mu, as described in EP 0332448, or phage lambda (λ). Thecopy number can also be increased by incorporating an additional copyinto another site in the chromosome, for example in to the att site ofphage λ (Yu and Court, Gene 223, 77-81 (1998)). U.S. Pat. No. 5,939,307reports that it was possible to increase the expression by incorporatingexpression cassettes or promoters, such as the tac promoter, the trppromoter, the lpp promoter, or the P_(L) promoter or P_(R) promoter ofphage λ, upstream, for example, of the chromosomal threonine operon. Inthe same way, it is possible to use the phage T7 promoters, the gearboxpromoters or the nar promoter. Such expression cassettes or promoterscan also be used, as described in EP 0 593 792, to overexpressplasmid-bound genes. Using the lacI^(Q) allele in turn makes it possibleto control the expression of plasmid-bound genes (Glascock and Weickert,Gene 223, 221-231 (1998)). It is furthermore possible for the activityof the promoters to be increased by modifying their sequence by means ofone or more nucleotide substitutions, by means of (an) insertion(s)and/or by means of (a) deletion(s). A different chronological geneexpression can be achieved, for example, as described in Walker et al.(Journal of Bacteriology 181: 1269-80 (1999)), by using the growthphase-dependent fis promoter. The rate of elongation is influenced bythe codon usage; gene expression can be enhanced by using codons fortRNAs which occur frequently in the parent strain.

The skilled person can find general instructions in this regard in,inter alia, Chang and Cohen (Journal of Bacteriology 134: 1141-1156(1978)), Hartley and Gregori (Gene 13: 347-353 (1981)), Amann andBrosius (Gene 40: 183-190 (1985)), de Broer et al. (Proceedings of theNational Academy of Sciences of the United States of America 80: 21-25(1983)), LaVallie et al. (BIO/TECHNOLOGY 11: 187-193 (1993)), inPCT/US97/13359, Llosa et al. (Plasmid 26: 222-224 (1991)), Quandt andKlipp (Gene 80: 161-169 (1989)), Hamilton et al. (Journal ofBacteriology 171: 4617-4622 (1989)), Jensen and Hammer (Biotechnologyand Bioengineering 58: 191-195 (1998)) and known textbooks of geneticsand molecular biology.

Plasmid vectors which can be replicated in Enterobacteriaceae, such aspACYC184-derived cloning vectors (Bartolomé et al.; Gene 102: 75-78(1991)), pTrc99A (Amann et al.; Gene 69: 301-315 (1988)) or pSC101derivatives (Vocke and Bastia; Proceedings of the National Academy ofSciences USA 80(21): 6557-6561 (1983)) can be used. In a processaccording to the invention, it is possible to use a strain which istransformed with a plasmid vector which carries at least the yjcG-ORF,or nucleotide sequences, or alleles, which encode its gene product.

The term “transformation” is understood as meaning the uptake of anisolated nucleic acid by a host (microorganism).

It is also possible to use sequence exchange (Hamilton et al.; Journalof Bacteriology 171: 4617-4622 (1989)), conjugation or transduction totransfer mutations, which affect the expression of the given genes oropen reading frames, into different strains.

More detailed explanations of the concepts of genetics and molecularbiology can be found in known textbooks of genetics and molecularbiology such as the textbook by Birge (Bacterial and BacteriophageGenetics, 4^(th) ed., Springer Verlag, New York (USA), 2000) or thetextbook by Berg, Tymoczko and Stryer (Biochemistry, 5^(th) ed., Freemanand Company, New York (USA), 2002) or the manual by Sambrook et al.(Molecular Cloning, A Laboratory Manual, (3-Volume Set), Cold SpringHarbor Laboratory Press, Cold Spring Harbor (USA), 2001).

Furthermore, when using strains of the Enterobacteriaceae family toproduce L-amino acids, in particular L-threonine, it can beadvantageous, in addition to potentiating the open reading frame yjcG,to enhance one or more enzymes of the known threonine biosynthesispathway or enzymes of anaplerotic metabolism or enzymes for producingreduced nicotinamide adenine dinucleotide phosphate or enzymes ofglycolysis or PTS enzymes or enzymes of sulfur metabolism. Usingendogenous genes is generally preferred.

Thus, it is possible, for example, to simultaneously enhance, inparticular overexpress, one or more of the genes selected from the group

-   -   at least one gene of the thrABC operon encoding aspartate        kinase, homoserine dehydrogenase, homoserine kinase and        threonine synthase (U.S. Pat. No. 4,278,765),    -   the pyruvate carboxylase-encoding Corynebacterium glutamicum pyc        gene (WO 99/18228),    -   the phosphoenolpyruvate synthase-encoding pps gene (Molecular        and General Genetics 231(2): 332-336 (1992); WO 97/08333),    -   the phosphoenolpyruvate carboxylase-encoding ppc gene (WO        02/064808),    -   the pntA and pntB genes encoding the subunits of        transhydrogenase (European Journal of Biochemistry 158: 647-653        (1986); WO 95/11985),    -   the rhtC gene encoding the threonine resistance-mediating        protein (EP-A-1 013 765),    -   the threonine export carrier protein-encoding Corynebacterium        glutamicum thrE gene (WO 01/92545),    -   the glutamate dehydrogenase-encoding gdhA gene (Nucleic Acids        Research 11: 5257-5266 (1983); Gene 23: 199-209 (1983);        DE19907347),    -   the ptsHIcrr operon ptsH gene encoding the phospho-histidine        protein hexose phosphotransferase of the PTS phosphotransferase        system (WO 03/004674),    -   the ptsHIcrr operon ptsI gene encoding enzyme I of the PTS        phosphotransferase system (WO 03/004674),    -   the ptsHIcrr operon crr gene encoding the glucose-specific IIA        component of the PTS phosphotransferase system (WO 03/004674),    -   the ptsG gene encoding the glucose-specific IIBC component (WO        03/004670),    -   the cysteine synthase A-encoding cysK gene (WO 03/006666),    -   the cysB gene encoding the regulator of the cys regulon (WO        03/006666),    -   the cysJIH operon cysJ gene encoding the NADPH sulfite reductase        flavoprotein (WO 03/006666),    -   the cysJIH operon cysI gene encoding the NADPH sulfite reductase        hemoprotein (WO 03/006666),    -   the adenylyl sulfate reductase-encoding cysJIH operon cysH gene        (WO 03/006666),    -   the sucABCD operon sucA gene encoding the decarboxylase subunit        of 2-ketoglutarate dehydrogenase (WO 03/008614),    -   the sucABCD operon sucB gene encoding the        dihydrolipoyl-transsuccinase E2 subunit of 2-ketoglutarate        dehydrogenase (WO 03/008614),    -   the suc ABCD operon sucC gene encoding the β-subunit of        succinyl-CoA synthetase (WO 03/008615),    -   the sucABCD operon sucD gene encoding the α-subunit of        succinyl-CoA synthetase (WO 03/008615),    -   the gene product of the Escherichia coli yibD open reading frame        (ORF) (Accession Number AE000439 of the National Center for        Biotechnology Information (NCBI, Bethesda, Md., USA,        DE102004005836.9)), and    -   the gene acs encoding the acetyl coenzyme A synthetase (Journal        of Bacteriology 177(10): 2878-86 (1995)).

Furthermore, for the purpose of producing L-amino acids, in particularL-threonine, it can be advantageous, in addition to enhancing the openreading frame yjcG, to attenuate, in particular eliminate or reduce theexpression of one or more of the genes selected from the group

-   -   the threonine dehydrogenase-encoding tdh gene (Journal of        Bacteriology 169: 4716-4721 (1987)),    -   the malate dehydrogenase (E.C. 1.1.1.37)-encoding mdh gene        (Archives in Microbiology 149: 36-42 (1987)),    -   the gene product of the Escherichia coli yjfA open reading frame        (ORF) (Accession Number AAC77180 of the National Center for        Biotechnology Information (NCBI, Bethesda, Md., USA), (WO        02/29080),    -   the gene product of the Escherichia coli ytfP open reading frame        (ORF) (Accession Number AAC77179 of the National Center for        Biotechnology Information (NCBI, Bethesda, Md., USA), WO        02/29080)),    -   the pckA gene encoding the enzyme phosphoenolpyruvate        carboxykinase (WO 02/29080),    -   the pyruvate oxidase-encoding poxB gene (WO 02/36797),    -   the dgsA gene (WO 02/081721), which is also known under the name        mlc gene, encoding the DgsA regulator of the phosphotransferase        system,    -   the fruR gene (WO 02/081698), which is also known under the name        cra gene, encoding the fructose repressor,    -   the rpoS gene (WO 01/05939), which is also known under the name        katF gene, encoding the sigma³⁸ factor, and    -   the aspartate ammonium lyase-encoding aspA gene (WO 03/008603).

In this context, the term “attenuation” describes the reduction orabolition, in a microorganism, of the intra-cellular activity orconcentration of one or more enzymes or proteins which are encoded bythe corresponding DNA, by, for example, using a weaker promoter than inthe parent strain or microorganism not recombinant for the correspondingenzyme or protein, or a gene or allele which encodes a correspondingenzyme or protein having a lower activity, or inactivating thecorresponding enzyme or protein, or the open reading frame or gene, and,where appropriate, combining these measures.

In general, the attenuation measures lower the activity or concentrationof the corresponding protein down to from 0 to 75%, from 0 to 50%, from0 to 25%, from 0 to 10% or from 0 to 5% of the activity or concentrationof the wild-type protein or of the activity or concentration of theprotein for the parent strain or microorganism which is not recombinantfor the corresponding enzyme or protein. The parent strain ormicroorganism which is not recombinant is understood as being themicroorganism on which the measures according to the invention areperformed.

In order to achieve an attenuation, for example the expression of thegenes or open reading frames, or the catalytic properties of the enzymeproteins, can be reduced or abolished. Where appropriate, both measurescan be combined.

The gene expression can be reduced by carrying out the culture in asuitable manner, by genetically altering (mutating) the signalstructures for the gene expression or by means of the antisense RNAtechnique. Signal structures for the gene expression are, for example,repressor genes, activator genes, operators, promoters, attenuators,ribosome binding sites, the start codon and terminators. The skilledperson can find information in this regard in, inter alia and forexample, Jensen and Hammer (Biotechnology and Bioengineering 58: 191-195(1998)), in Carrier and Keasling (Biotechnology Progress 15: 58-64(1999)), in Franch and Gerdes (Current Opinion in Microbiology 3:159-164 (2000)) and in well known textbooks of genetics and molecularbiology such as the textbook by Knippers (“Molekulare Genetik [MolecularGenetics]”, 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995)or that by Winnacker (“Gene und Klone [Genes and Clones]”, VCHVerlagsgesellschaft, Weinheim, Germany, 1990).

Mutations which lead to a change or reduction in the catalyticproperties of enzyme proteins are known from the prior art. Exampleswhich may be mentioned are the articles by Qiu and Goodman (Journal ofBiological Chemistry 272: 8611-8617 (1997)), Yano et al. (Proceedings ofthe National Academy of Sciences of the United States of America 95:5511-5515 (1998)) and Wente and Schachmann (Journal of BiologicalChemistry 266: 20833-20839 (1991)). Summaries can be found in knowntextbooks of genetics and molecular biology, such as that by Hagemann(“Allgemeine Genetik [General Genetics]”, Gustav Fischer Verlag,Stuttgart, 1986).

Mutations which come into consideration are transitions, transversions,insertions and deletions of at least one (1) base pair or nucleotide.Depending on the effect of the mutation-elicited amino acid substitutionon the enzyme activity, reference is made to missense mutations or tononsense mutations. A missense mutation leads to the replacement of agiven amino acid in a protein with a different amino acid, with theamino acid replacement in particular being non-conservative. Thisthereby impairs the functional ability or activity of the protein andreduces it down to a value of from 0 to 75%, 0 to 50%, 0 to 25%, 0 to10% or 0 to 5%. A nonsense mutation leads to a stop codon in the codingregion of the gene and thus to premature termination of the translation.Insertions or deletions of at least one base pair in a gene lead toframe shift mutations which in turn result in incorrect amino acidsbeing incorporated or in the translation being prematurely terminated.If a stop codon is formed in the coding region as a consequence of themutation, this then also leads to translation being terminatedprematurely.

Deletions of at least one (1) or more codons typically also lead tocomplete loss of the enzyme activity. Directions for generating thesemutations belong to the prior art and can be obtained from knowntextbooks of genetics and molecular biology such as the textbook byKnippers (“Molekulare Genetik [Molecular Genetics]”, 6th edition, GeorgThieme Verlag, Stuttgart, Germany, 1995), that by Winnacker “Gene undKlone, [Genes and Clones]”, VHC Verlagsgesellschaft, Weinheim, Germany,1990) or that by Hagemann (“Allgemeine Genetik [General Genetics]”,Gustav Fischer Verlag, Stuttgart, 1986).

Suitable mutations in the genes can be incorporated into suitablestrains by means of gene or allele exchange. A customary method is themethod, described by Hamilton et al. (Journal of Bacteriology 171:4617-4622 (1989)), of gene exchange using a conditionally replicatingpSC101 derivative pMAK705. Other methods described in the prior art,such as that of Martinez-Morales et al. (Journal of Bacteriology 181:7143-7148 (1999)) or that of Boyd et al. (Journal of Bacteriology 182:842-847 (2000)), can also be used.

It is likewise possible to transfer mutations in the relevant genes, ormutations which effect the expression of the relevant genes or openreading frames, into different strains by means of conjugation ortransduction.

Furthermore, for the purpose of producing L-amino acids, in particularL-threonine, it can be advantageous, in addition to enhancing the openreading frame yjcG, to eliminate undesirable side-reactions (Nakayama:“Breeding of Amino Acid Producing Microorganisms”, in: Overproduction ofMicrobial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press,London, UK, 1982).

The performance of the isolated bacteria, or of the fermentation processusing these bacteria, is improved, with regard to one or more of theparameters selected from the group consisting of the productconcentration (product per volume), the product yield (product formedper carbon source consumed) and the product formation (product formedper volume and time), or else other process parameters and combinationsthereof, by at least 0.5%, at least 1%, at least 1.5% or at least 2%,based on the nonrecombinant microorganism or parent strain, or thefermentation process using this microorganism or parent strain.

The microorganisms which are prepared in accordance with the inventioncan be cultured in a batch process, in a fed-batch process, in arepeated fed-batch process or in a continuous process (DE102004028859.3or U.S. Pat. No. 5,763,230). Known culturing methods are summarized inthe textbook by Chmiel (Bioprozesstechnik 1. Einführung in dieBioverfahrenstechnik [Bioprocess technology 1. Introduction tobioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991)) or inthe textbook by Storhas (Bioreaktoren und periphere Einrichtungen[Bioreactors and peripheral installations] (Vieweg Verlag,Brunswick/Wiesbaden, 1994)).

The culture medium to be used must satisfy the demands of the givenstrains in an appropriate manner. The American Society for Bacteriologymanual “Manual of Methods for General Bacteriology” (Washington D.C.,USA, 1981) contains descriptions of media for culturing a variety ofmicroorganisms.

Sugars and carbohydrates, such as glucose, sucrose, lactose, fructose,maltose, molasses, starch and, where appropriate, cellulose, oils andfats, such as soybean oil, sunflower oil, peanut oil and coconut fat,fatty acids, such as palmitic acid, stearic acid and linoleic acid,alcohols, such as glycerol and ethanol, and organic acids, such asacetic acid, may be used as the carbon source. These substances may beused individually or as a mixture. For example, mixtures of glucose andfructose can be used in a ratio of approx. 1:1, as described in EP 1 225230.

Organic nitrogen-containing compounds, such as peptones, yeast extract,meat extract, malt extract, corn steep liquor, soybean flour and urea,or inorganic compounds, such as ammonium sulfate, ammonium chloride,ammonium phosphate, ammonium carbonate and ammonium nitrate, may be usedas the nitrogen source. The nitrogen sources may be used individually oras a mixture.

Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogenphosphate, or the corresponding sodium-containing salts, may be used asthe phosphorus source. In addition, the culture medium must containsalts of metals, such as magnesium sulfate or iron sulfate, which arerequired for growth. Finally, essential growth promoters, such as aminoacids and vitamins, may be used in addition to the abovementionedsubstances. Suitable precursors can also be added to the culture medium.Said ingredients may be added to the culture in the form of a one-offmixture or suitably fed in during the culture.

The fermentation is generally carried out at a pH of from 5.5 to 9.0, inparticular of from 6.0 to 8.0. Basic compounds, such as sodiumhydroxide, potassium hydroxide, ammonia or ammonia water, or acidiccompounds, such as phosphoric acid or sulfuric acid, are used in asuitable manner for controlling the pH of the culture. Antifoamants,such as fatty acid polyglycol esters, can be used for controllingfoaming. Suitable selectively acting substances, for exampleantibiotics, can be added to the medium in order to maintain thestability of plasmids. Oxygen or oxygen-containing gas mixtures, such asair, are passed into the culture in order to maintain aerobicconditions. The temperature of the culture is normally from 25° C. to45° C. and preferably from 30° C. to 40° C. The action of themicroorganisms results in the L-amino acid being accumulated in theculture broth. The culture is continued until a maximum of L-amino acidsor L-threonine has been formed. This objective is normally reachedwithin 10 to 160 hours.

The L-amino acids can be isolated, collected or concentrated from theculture broth, which has been taken off, and then purified, whereappropriate. Ion exchange chromatography and crystallization are typicalmethods for purifying the L-amino acids. These methods result in L-aminoacids which are to a large extent pure.

It is likewise possible to prepare a product from the culture broth(=fermentation broth), which has been taken off, by removing the biomassof the bacterium, which is present in the culture broth, completely(100%) or almost completely, i.e. more than or greater than (>) 90%, andto a large extent, i.e. to an extent of 30%-100%, preferably greaterthan or equal to (≧) 50%, ≧70% or ≧90%, or else completely (100%),leaving the remaining constituents of the fermentation broth in theproduct.

Separation methods such as centrifugation, filtration, decantation orflocculation, or a combination thereof, are used for removing orseparating off the biomass.

The resulting broth is then inspissated or concentrated using knownmethods, for example using a rotary evaporator, a thin film evaporatoror a falling film evaporator, by means of reverse osmosis or by means ofnanofiltration, or a combination of these methods.

This concentrated broth is then worked-up into what is preferably aflowable, finely divided powder using the methods of freeze drying,spray drying or spray granulation, or using other methods. Thisflowable, finely divided powder can then in turn be converted into acoarse-grain, readily flowable, storable, and to a large extentdust-free, product using suitable compacting or granulating methods. Atotal of more than 90% of the water is removed in this connection, suchthat the water content in the product is less than 10%, less than 5% orless than 3%.

L-amino acids can be analyzed by means of anion exchange chromatographyfollowed by derivatization with ninhydrin, as described in Spackman etal. (Analytical Chemistry 30: 1190-1206 (1958)), or by means of reversedphase HPLC, so as described in Lindroth et al. (Analytical Chemistry 51:1167-1174 (1979)).

The process according to the invention can be used for fermentativelypreparing L-amino acids, such as L-threonine, L-isoleucine, L-valine,L-methionine, L-homoserine, L-tryptophan and L-lysine, in particularL-threonine.

The present invention is explained in more detail below with the aid ofimplementation examples.

Minimal (M9) and complete (LB) media used for Escherichia coli aredescribed by J. H. Miller (A short course in bacterial genetics (1992),Cold Spring Harbor Laboratory Press). The isolation of plasmid DNA fromEscherichia coli, and also all techniques for restricting, ligating andtreating with Klenow phosphatase and alkali phosphatase, are carried outas described in Sambrook et al. (Molecular Cloning—A Laboratory Manual(1989) Cold Spring Harbor Laboratory Press). Unless otherwise indicated,Escherichia coli are transformed as described in Chung et al.(Proceedings of the National Academy of Sciences of the United States ofAmerica 86: 2172-2175 (1989)).

The incubation temperature when preparing strains and transformants is37° C.

EXAMPLE 1

Constructing the Expression Plasmid pMW218yjcG

The E. coli K12 yjcG-ORF is amplified using the polymerase chainreaction (PCR) and synthetic oligonucleotides. PCR primers aresynthesized (MWG Biotech, Ebersberg, Deutschland) on the basis of thenucleotide sequence of the yjcG gene in E. coli K12 MG1655 (AccessionNumber NC000913 (Region 4281276-4282925), Blattner et al. (Science 277:1453-1474 (1997)). The sequences of the primers are modified so as toform recognition sites for restriction enzymes. The HindIII recognitionsequence is selected for the yjcG-1 primer and the SacI recognitionsequence is selected for the yjcG-2 primer, with these sequences beingunderlined in the nucleotide sequences shown below: yjcG-1: (SEQ ID No.7) 5′-GATCAAGCTTATCCGGCCTACATTCG-3′

yjcg-2: (SEQ ID No. 8) 5′-GATCTAGAGCTCGATTAATGCGCGCGGCCTT-3′

The E. coli K12 MG1655 chromosomal DNA used for the PCR is isolatedusing “Qiagen Genomic-tips 100/G” (QIAGEN, Hilden, Germany) inaccordance with the manufacturer's instructions. A DNA fragment ofapprox. 2084 bp in size (SEQ ID No. 9) can be amplified under standardPCR conditions (Innis et al. (1990) PCR Protocols. A Guide to Methodsand Applications, Academic Press) using Vent DNA polymerase (New EnglandBiolaps GmbH, Frankfurt, Germany) and the specific primers.

The amplified yjcG fragment is ligated to the vector pCR-Blunt II-TOPO(Zero TOPO TA Cloning Kit, Invitrogen, Groningen, Netherlands) inaccordance with the manufacturers instructions and transformed into theE. coli strain TOP10. Plasmid-harboring cells are selected on LB Agarcontaining 50 μg of kanamycin/ml. After the plasmid DNA has beenisolated, the vector is cleaved with the enzymes PvuI and HindIII/SacIand, after the cleavage has been checked in a 0.8% agarose gel,designated pCRBluntyjcG.

The vector pCRBluntyjcG is then cleaved with the enzymes HindIII andSacI and the yjcG fragment is separated in a 0.8% agarose gel; it isthen isolated from the gel (QIAquick Gel Extraction Kit, QIAGEN, Hilden,Germany) and ligated to the low-copy vector pMW218 (Nippon Gene, Toyama,Japan) which has been digested with the enzymes HindIII and SacI. The E.coli strain DH5α (Grant et al.; Proceedings of the National Academy ofSciences USA, 87 (1990) 4645-4649) is transformed with the ligationmixture and plasmid-harboring cells are selected on LB agar containing50 μg of kanamycin/ml.

The fact that cloning has been successful can be demonstrated, after theplasmid DNA has been isolated, by performing a control cleavage usingthe enzymes EcoRI/SalI.

The plasmid is designated pMW218yjcG (FIG. 1).

EXAMPLE 2

Preparing L-Threonine Using the Strain MG442/pMW218yjcG

The L-threonine-producing E. coli strain MG442 is described in thepatent specification U.S. Pat. No. 4,278,765 and is deposited in theRussian national collection of industrial microorganisms (VKPM, Moscow,Russia) as CMIM B-1628.

The strain MG442 is transformed with the expression plasmid pMW218yjcGdescribed in example 1, and with the vector pMW218, andplasmid-harboring cells are selected on LB agar containing 50 μg ofkanamycin/ml. This results in the strains MG442/pMW218yjcG andMG442/pMW218. Selected individual colonies are then propagated furtheron minimal medium having the following composition: 3.5 g ofNa₂HPO₄*2H₂O/l, 1.5 g of KH₂PO₄/l, 1 g of NH₄Cl/l, 0.1 g ofMgSO₄*7H₂O/l, 2 g of glucose/l, 20 g of agar/l, 50 mg of kanamycin/l.The formation of L-threonine is checked in 10 ml batch cultures whichare contained in 100 ml Erlenmeyer flasks. For this, a 10 ml preculturemedium of the following composition: 2 g of yeast extract/l, 10 g of(NH₄)₂SO₄/l, 1 g of KH₂PO₄/l, 0.5 g of MgSO₄*7H₂O/l, 15 g of CaCO₃/l, 20g of glucose/l, 50 mg of kanamycin/l, is inoculated and incubated, at37° C. and 180 rpm for 16 hours, on a Kühner AG ESR incubator(Birsfelden, Switzerland). In each case 250 μl of this preliminaryculture are inoculated over into 10 ml of production medium (25 g of(NH₄)₂SO₄/l, 2 g of KH₂PO₄/l, 1 g of MgSO₄*7H₂O/l, 0.03 g ofFeSO₄*7H₂O/l, 0.018 g of MnSO₄*1H₂O/l, 30 g of CaCO₃/l, 20 g ofglucose/l, 50 mg of kanamycin/l) and incubated at 37° C. for 48 hours.After the incubation, the optical density (OD) of the culture suspensionis determined at a measurement wavelength of 660 nm using a Dr. LangeLP2W photometer (Düsseldorf, Germany).

An Eppendorf-BioTronik amino acid analyzer (Hamburg, Germany) is thenused to determine, by means of ion exchange chromatography andpost-column reaction involving ninhydrin detection, the concentration ofthe resulting L-threonine in the culture supernatant, which has beensterilized by filtration.

The result of the experiment is shown in table 1. TABLE 1 OD Strain (660nm) L-Threonin g/l MG442/pMW218 6.4 2.15 MG442/pMW218yjcG 5.5 2.5

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Map of the yjcG gene-containing plasmid MW218yjcG

Length specifications are to be regarded as being approximate. Theabbreviations and designations employed have the following meanings:

-   -   kan: gene which encodes resistance to kanamycin    -   yjcG: coding region of the yjcG gene    -   lacZ′: gene fragment which encodes the α-peptide of        β-galactosidase

The abbreviations for the restriction enzymes have the followingmeaning:

-   -   EcoRI: restriction endonuclease from Escherichia coli    -   HindIII: restriction endonuclease from Haemophilus influenzae    -   SacI: restriction endonuclease from Streptomyces achromogenes    -   SalI: restriction endonuclease from Streptonyces albus

1. A recombinant microorganism which contains an enhanced oroverexpressed yjcG-ORF, the gene product of which has acetate permeaseactivity.
 2. A microorganism as claimed in claim 1, in which apolynucleotide which corresponds to yjcG-ORF and encodes a polypeptidewhose amino acid sequence is at least 90% identical to an amino acidsequence selected from the group SEQ ID No. 2, SEQ ID No. 4 and SEQ IDNo. 6 is enhanced, the polypeptide having acetate permease activity. 3.A microorganism as claimed in claim 2, characterized in that it containsan overexpressed or enhanced polynucleotide which corresponds toycjG-ORF and which is selected from the group: a) polynucleotide havinga nucleotide sequence, selected from SEQ ID No. 1, SEQ ID No. 5 and SEQID No. 3 and the nucleotide sequences complementary thereto; b)polynucleotide having a nucleotide sequence which corresponds to SEQ IDNo. 1, SEQ ID No. 3 or SEQ ID No. 5 within the limits of the degeneracyof the genetic code; c) polynucleotide sequence having a sequence whichhybridizes, under stringent conditions, with the sequence which iscomplementary to the sequence SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No.5, with the stringent conditions being achieved by means of a washingstep in which the temperature extends over a range of from 64° C. to 68°C. and the salt concentration of the buffer extends over a range of from2×SSC to 0.1×SSC; d) polynucleotide having a sequence SEQ ID No. 1, SEQID No. 3 or SEQ ID No. 5 which contains functionally neutral sensemutants.
 4. A microorganism as claimed in claim 2, characterized in thatthe polypeptide possesses an amino acid sequence which is at least 95%identical to one of the sequences selected from the group SEQ ID No. 2,SEQ ID No. 4 and SEQ ID No.
 6. 5. A microorganism as claimed in claim 2,characterized in that the polypeptide possesses the amino acid sequencewhich is 100% identical to that of one of the sequences selected fromthe group consisting of SEQ ID No. 2, SEQ ID No. 4 and SEQ ID No.6.
 6. Amicroorganism as claimed in claim 1, characterized in that it isproduced by transformation, transduction or conjugation, or acombination of these methods, with a vector which contains the yjcG-ORF,an allele of this ORF, or parts thereof, and/or a promotor.
 7. Amicroorganism as claimed in claim 1, in which the copy number of theyjcG-ORF or the alleles has been increased by at least
 1. 8. Themicroorganism as claimed in claim 7, characterized in that the increasein the copy number of the yjcG-ORF by at least 1 is achieved byintegrating the ORF or the alleles into the chromosome of themicroorganism.
 9. The microorganism as claimed in claim 7, characterizedin that the increase in the copy number of the yjcG-ORF by at least 1 isachieved by means of a vector which replicates extrachromosomally. 10.The microorganism as claimed in claim 1, characterized in that, in orderto achieve the enhancement, a) the promoter and regulatory region or theribosomal binding site upstream of the yjcG-ORF is mutated, or b)expression cassettes or promoters are incorporated upstream of theyjcG-ORF.
 11. The microorganism as claimed in claim 1, characterized inthat the expression of the yjcG-ORF is under the control of a promoterenhancing the expression of the ORF.
 12. The microorganism as claimed inclaim 1, characterized in that enhancing the yjcG-ORF increases theconcentration or activity of the yjcG gene product (protein) by at least10%, based on the activity or concentration of the gene product in theparent strain or microorganism not recombinant for the yjcG-ORF.
 13. Themicroorganism as claimed in claim 1, characterized in that themicroorganism is selected from the genera Escherichia, Erwinia,Providencia and Serratia.
 14. The microorganism as claimed in claim 13,characterized in that other genes of the pathway for the biosynthesis ofthe desired L-amino acid are also present in enhanced, in particularoverexpressed, form.
 15. The microorganism as claimed in claim 1,characterized in that it produces L-threonine.
 16. A process forpreparing L-amino acids by fermenting recombinant microorganisms of theEnterobacteriaceae family, characterized in that a) the desired L-aminoacid-producing microorganisms as claimed in claim 1 are cultured in amedium under conditions under which the desired L-amino acid isaccumulated in the medium or in the cells, and b) the desired L-aminoacid is isolated, with constituents of the fermentation broth, and/orthe biomass remaining in its/their entirety or in portions (from ≧0 to100%) in the isolated product or being removed completely.
 17. Theprocess as claimed in claim 16, characterized in that, for the purposeof preparing L-threonine, microorganisms are fermented in which one ormore of the genes selected from the group: a) at least one gene of thethrABC operon encoding aspartate kinase, homoserine dehydrogenase,homoserine kinase and threonine synthase, b) the pyruvatecarboxylase-encoding Corynebacterium glutamicum pyc gene, c) thephosphoenolpyruvate synthase-encoding pps gene, d) thephosphoenolpyruvate carboxylase-encoding ppc gene, e) the pntA and pntBgenes encoding the subunits of pyridine transhydrogenase, f) the rhtCgene encoding the threonine resistance-mediating protein, g) thethreonine export carrier protein-encoding Corynebacterium glutamicumthrE gene, h) the glutamate dehydrogenase-encoding gdhA gene, i) theptsH gene encoding the phosphohistidine protein hexosephosphotransferase, j) the ptsI gene encoding enzyme I of thephosphotransferase system, k) the crr gene encoding the glucose-specificIIA component, l) the ptsG gene encoding the glucose-specific IIBCcomponent, m) the cysteine synthase A-encoding cysK gene, n) the cysBgene encoding the regulator of the cys regulon, o) the cysJ geneencoding the NADPH sulfite reductase flavoprotein, p) the cysI geneencoding the NADPH sulfite reductase hemoprotein, q) the adenylylsulfate reductase-encoding cysH gene, r) the sucA gene encoding thedecarboxylase subunit of 2-ketoglutarate dehydrogenase, s) the sucB geneencoding the dihydrolipoyl-transsuccinase E2 subunit of 2-ketoglutaratedehydrogenase, t) the sucC gene encoding the β-subunit of succinyl-CoAsynthetase, u) the sucD gene encoding the α-subunit of succinyl-CoAsynthetase, v) the gene product of the Escherichia coli yibD openreading frame (ORF), and w) the acs encoding the acetyl coenzyme Asynthetase is/are additionally, at the same time, enhanced, inparticular overexpressed.
 18. The process as claimed in claim 16,characterized in that use is made of microorganisms in which themetabolic pathways which reduce the formation of the desired L-aminoacid are at least partially attenuated.
 19. The process as claimed inclaim 18, characterized in that, for the purpose of preparingL-threonine, microorganisms are fermented in which one or more of thegenes selected from the group: a) the threonine dehydrogenase-encodingtdh gene, b) the malate dehydrogenase-encoding mdh gene, c) the geneproduct of the Escherichia coli yjfA open reading frame (ORF), d) thegene product of the Escherichia coli ytfP open reading frame (ORF), e)the pckA gene encoding the phosphoenolpyruvate carboxykinase, f) thepyruvate oxidase-encoding poxB gene, g) the dgsA gene encoding the DgsAregulator of the phosphotransferase system, h) the fruR gene encodingthe fructose repressor, i) the rpoS gene encoding the sigma³⁸ factor,and, j) the aspartate ammonium lyase-encoding aspA gene, is/areadditionally, at the same time, attenuated, in particular eliminated, ortheir expression is reduced.
 20. The process as claimed in claim 16,characterized in that L-amino acids selected from the groupL-asparagine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine,L-valine, L-methionine, L-proline, L-isoleucine, L-leucine, L-tyrosine,L-phenylalanine, L-histidine, L-lysine, L-tryptophan, L-arginine andL-homoserine are prepared.
 21. The process as claimed in claim 16,characterized in that L-amino acids selected from the groupL-isoleucine, L-valine, L-methionine, L-homoserine, L-tryptophan andL-lysine are prepared.